Assessing the role of eosinophil‐mediated immune response markers in detecting hookworm infection: A case‐control study in Kintampo, Ghana

Abstract Background and Aim Human hookworm disease caused by Ancylostoma duodenale and Necator americanus is a serious public health problem. Hookworm infection activates eosinophil‐mediated tissue inflammatory responses, involving the production of the eosinophil‐specific chemokine (eotaxin), recruitment of eosinophils, secretion of the cationic protein, and production of antiparasite immunoglobulin E (IgE). We investigated eosinophil‐mediated immune response as markers (CCL11, eosinophil cationic protein [ECP], and IgE) for detecting hookworm infection. Methods This case‐control study was carried out in hookworm endemic areas within the Kintampo North Municipality.Forty hookworm‐positive subjects and 36 apparently healthy individuals were recruited as cases and controls, respectively. Stool samples were collected for hookworm detection by the Kato–Katz technique and speciation by polymerase chain reaction. Approximately, 5 ml of intravenous blood was used to obtain plasma for the immunological assays. Results Of eosinophil‐mediated immune response markers studied, ECP and CCL11 were significantly higher among hookworm patients compared to controls. Increasing CCL11 (β = −0.81, p = 0.015) was associated with a significant decrease hookworm intensity. However, increasing eosinophil count (β = 0.62, p = 0.027) was associated with significant increase in hookworm intensity. In receiver operator characteristics analysis, ECP could significantly detect hookworm infection with a very high area under the curve (AUC) (AUC = 0.97, p < 0.0001). At a cutoff of 39.05, ECP was the best eosinophil‐mediated immune response marker for detecting hookworm infection with a sensitivity of 97.2%, specificity of 87.8%, a positive predictive value of 89.7%, and a negative predictive value of 96.6%. Conclusion ECP best predicts eosinophil‐mediated immune response for detecting hookworm infection, while CCL11 and eosinophil count better predict the intensity of hookworm. Moreover, the ECP level is a good indicator of hookworm infection and intensity and may require additional investigations to augment current hookworm diagnostic techniques.

and intensity and may require additional investigations to augment current hookworm diagnostic techniques. people. 1,2 To reduce soil-transmitted helminthiases, a thorough and well-timed review of epidemiological studies using sensitive and precise diagnostic techniques is needed. Lustigman et al. 3 suggested that implementation of interventions, monitoring, and evaluating their effectiveness will help in detecting anthelminthic resistance at the early stage. [3][4][5] Hookworm infection activates eosinophil-mediated tissue inflammatory responses and involves the production of the eosinophilspecific chemokine (eotaxin), recruitment of eosinophils, secretion of eosinophil cationic protein (ECP), and the secretion of anti-parasite immunoglobulin E (IgE). 6,7 The synergistic effect of these mediators protect the host against the hookworm parasites. 8,9 Eosinophils are granulocytic leukocytes, which duty is in host defense and tissue pathogenesis triggered by helminth infection. 10 In parasitic infections, eosinophils are increased and recruited into inflamed tissues under the guidance of Th2 cell-derived cytokines and chemokines. 11 These Th2 immune responses, culminate in IgE production and eosinophilia. IgE has been linked with protection against an extensive range of helminth infections and believed that IgE and its receptors support counter metazoan parasites. 8 Eosinophils possess many cell surface receptors for cell signaling linked with a respiratory burst, chemotaxis, adhesion, degranulation, or apoptosis, 12  Eotaxin (CCL11) is a vital specific eosinophil chemokine linked with the accumulation of eosinophils at sites of infection. It is also produced in the lungs of asthmatics and functions in directing eosinophils at inflammatory sites. 13 In addition, eotaxin is involved in the discriminatory recruitment of eosinophils into inflammatory areas in the course of parasitic and allergic reactions. Similarly, most soiltransmitted helminthiases are driven by type-2 (Th2). Ivanovska et al. 14 observed high levels of eotaxin in neurodegenerative and psychiatric conditions 15 ; however, no study has assessed whether eotaxin can be considered a marker for hookworm infection.
Eosinophil granulocytes produce an effective cytotoxic protein called the ECP which functions in host defense against helminth infections. 16 The presence of helminth or an allergen can lead to the release of ECP. 17 Subsequently, a high worm load can result in eosinophilia and hence a surge in circulating ECP levels. It is, thus, imperative to evaluate the diagnostic accuracy of ECP levels and its relation to egg counts in the milieu of the eosinophil-mediated immune response. 18 While the exploration for a cost-effective, sensitive, highly-specific, noninvasive diagnostic test for hookworm is imperative, it is important to study all products of eosinophilmediated immune response markers (CCL11, ECP, and IgE).
Studies directed at eosinophils have gained importance in recent times. However, no study has assessed products of eosinophil-linked tissue inflammatory reactions in hookworm infection. The current Kato-Katz method is saddled with low sensitivity, especially for the identification of low-intensity parasitic infections. 17,19 Against this background, the current study assessed products of eosinophilmediated immune responses (CCL11, ECP, and IgE) as potential diagnostic markers for hookworm infection. Identification of a sensitive correlation between these potential biomarkers and hookworm intensity will augment current diagnostic methods.

| Study site
The study was conducted in widespread communities within the

| Study design and sample processing
This case-control study was conducted in the KNM. Community engagement was done through a durbar to explain the purpose and the nature of the study. Consenting community members were all screened for parasitic infection, chronic, infectious, and allergic infections. Forty hookworm-positive subjects who fulfilled inclusion criteria were recruited as cases, while 36 apparently healthy individuals without any parasitic infection were recruited as controls.
Skilled field assistants administered structured health questionnaires, and shared labeled stool containers with the participants. Stool samples were obtained and processed for hookworm detection by the Kato-Katz technique and polymerase chain reaction(PCR). 20 Approximately 5 ml of blood was obtained by venepuncture into vacutainers containing ethylene diamine tetraacetic acid containers.
The blood sample was centrifuged, and the plasma was stored at −80°C until ready to be used.

| Crude N. americanus egg antigen preparation
Eggs isolated from stools of N. americanus infected individuals were used for the crude antigen extraction. The eggs were suspended in 4°C 1X phosphate-buffered saline (PBS) at a concentration of about 500 eggs/ml. A prechilled homogenizer was used to homogenize the eggs. The solution was boiled, frozen in liquid nitrogen, and thawed and homogenized three times. When approximately about 95% (or more) of the eggs were shredded/disrupted, the crude mixture was centrifuged at 4°C at 15,000 rpm for 60 min. The supernatant was collected and sterilized by passing it through a 0.2 µm filter. They were then aliquoted into 2 ml cryovial tubes and stored at −80°C.

| PCR identification of hookworm species
Hookworm species identification was determined using genomic DNA (gDNA) extracted from purified hookworm egg samples of infected individuals using QIAamp DNA Stool Kit (QIAGEN). 21,22 Purified gDNA (20-40 ng) was used in PCR for the amplification of the internal transcribed region of ribosomal DNA. 23 The PCR reaction Negative (no template, nuclease-free water) controls were included in all experiments. The PCR cycling conditions were, initial heating at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, with a final elongation step at 72°C for 5 min. The amplified products were visualized and the sizes were determined by UV visualization after electrophoresis in a 2% ethidium bromide stained-agarose gel.
Products of the appropriate size (690 bp for A. duodenale and 870 bp for N. americanus) were considered positive compared to standard controls.

| Blood eosinophil determination
The blood eosinophil levels were determined using a hematology analyzer (ABX Pentra 60C+; HORIBA Medical) by following the manufacturer's instructions.

| Statistical analyses
Statistical analyses were performed on the R language for statistical computing. 27 Age was presented as median with interquartile ranges for both groups whilst gender was presented as frequencies with percentages. Distribution and levels of eosinophil-mediated immune response markers were present by kernel density plot and violin plot respectively and subsequent Mann-Whitney U test. A multiple linear regression model was used to assess the association between eosinophil-mediated immune response markers and hookworm intensity. The receiver operator characteristics (ROC) analysis from the pROC package in R 28 was used to determine the diagnostic accuracies of the markers. p-values less than 0.05 were considered statistically significant.   (Figure 1).

| Association between eosinophil-mediated immune responses markers and hookworm intensity
In a multivariate linear regression model, increasing CCL11 (β = −0.81, p = 0.015) was associated with a significant decrease in hookworm intensity. Again, increasing IgE (β = −0.01, p = 0.978) was associated with a slight decrease in hookworm intensity; however, the association was not statistically significant. In contrast, increasing eosinophil count (β = 0.62, p = 0.027) was associated with significant increase in hookworm intensity. Moreover, increasing ECP (β = 0.62, p = 0.027) was associated with an increase in hookworm intensity; however, this association was not statistically significant ( Figure 2).

| DISCUSSION
The role of eosinophils and parasite-killing antibody isotype IgE has been described in the control of hookworm infections. 29 34 It may be that with increased hookworm intensity, increased eosinophil levels were not via a CCL11-induced pathway.
The interplay of various eotaxins in the advent of eosinophilia in parasitic helminth infections needs to be clarified. including ours, found that increasing plasma ECP correlates with increasing hookworm infection intensity. 18,20,31 Although the current study reported the same, this correlation did not reach the level of statistical significance.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this article and its Supporting Information files data and can be requested from the corresponding author.